Metabolic‐developmental crosstalk: BmDHFR‐mediated Hedgehog signaling orchestrates organ development and lipid homeostasis in Bombyx mori

Dihydrofolate reductase (DHFR), a pivotal enzyme in folate metabolism and DNA synthesis, is a well-established cancer therapeutic target. However, its non-canonical roles in developmental signaling and evolutionary-conserved functions in non-mammalian species remain poorly characterized. Using the lepidopteran model Bombyx mori, this study uncovered the pleiotropic functions of BmDHFR in midgut proliferation and lipid homeostasis through dual metabolic and Hedgehog (Hh) signaling regulation. Spatiotemporal profiling revealed ubiquitous BmDHFR expression during larval development. RNA interference-mediated BmDhfr knockdown suppressed midgut cell proliferation, validating its conserved role in DNA replication via thymidylate (deoxythymidine monophosphate [dTMP]) synthesis, while unexpectedly up-regulating the adipogenic marker BmAP2 and inducing lipid droplet hypertrophy-contrasting mammalian white adipocyte differentiation. Mechanistically, BmDHFR directly interacts with silkworm suppressor of fused (BmSUFU), the core suppressor of Hh signaling, forming a "metabolism-signaling-cell fate" axis that bridges folate metabolism to Hh pathway regulation. Cross-species comparisons highlight evolutionary divergence: Hh signaling inhibits insect adipogenesis but regulates mammalian adipocyte differentiation, suggesting adaptive remodeling of DHFR-Hh networks. This study advances the functional characterization of BmDHFR, elucidating evolutionary conservation and species-specific divergence in metabolic enzyme pleiotropy, and lays a foundation for drug screening and disease research based on insect models.